We have established the association of hepatitis C virus (HCV) infection with type II cryoglobulinemia (MC-II) and with the WA crossidiotype (XId) positive monoclonal rheumatoid factors (WA mRF), and the selective concentration of HCV and very low density lipoprotein (VLDL) in these cryoglobulins. We have also established that the LDL receptor mediates endocytosis of HCV and other members of the Flaviviridae family and that the apolipoprotein E epsilon2 allele in HCV infected patients increases the risk of developing MC three-fold. The broad, long-term objective of this proposal is to investigate how WA mRF are produced in MC-II and how they may affect chronic HCV infection. Two hypotheses will be tested: 1) In patients with MC-II associated with HCV infection, the WA mRF is produced as a result of chronic stimulation of B cells by complexes of HCV and VLDL. It is postulated from this hypothesis that initially a WA+ RF- IgM is produced and that rheumatoid factor activity arises as a result of a point mutation in the CDR3 with chronic HCV infection. It will be determined whether: a) WA+ RF- IgM has antibody activity to HCV VLDL, b) WA mRFs ve cross reactivity with the same antigen, and c) cells producing WA mRF- IgM are the precursors of those producing WA mRF+ IgM. Lymphoid aggregates in liver biopsies from HCV infected patients with mixed cryglobulinemia will be examined for the presence of WA+ RF- and WA+ RF+ B cells and the results compared to DNA and mRNA analysis for WA sequences from the same liver biopsies and paired peripheral bloods. 2) LDL receptor endocytosis is a major route of HCV infection of hepatocytes. The main physiologic role of WA antibodies is to block endocytosis of HCV VLDL complexes by the LDL receptors. The retarded endocytosis of HCV-VLDL complexes containing apolipoprotein E2 via the LDL receptor is the mechanism underlying the apo E2 risk factor for developing MC-II. Flow cytometry, in situ hybridization, and quantitative PCR assays will be used to study the endocytosis of HCV-lipoprotein complexes to determine a) the rate of endocytosis of HCV-VLDL of various apo E phenotypes and various HCV genotypes and b) the effect of WA mRF and WA+ RF-IgM on the rates of endocytosis. In addition, the role of lipoprotein concentration, apo E phenotypes and HCV genotypes on the distribution of HCV among lipoproteins in HCV infected individuals with and without cryoglobulinemia and on the selective concentration on VLDL with HCV in MC-II will be determined. The proposed studies may provide insights into the etiology of MC-II, the mechanism of HCV infection, and the role of natural antibody systems in the immune response to HCV, and may lead to better therapy, early detection and prophylaxis of the disease.